The present invention relates to a micromethod and means for the determination of steroids, in particular of 17.alpha.-hydroxy-progesterone, in human body liquids and to a new method and means for detection of congenital adrenal hyperplasia (CAH) in newborn humans.
CAH is an inborn error of metabolism which is transmitted by an autosomal recessive gene. This disorder is due to a 21-hydroxylase deficiency and is recognizable by a markedly increased blood level of 17.alpha.-hydroxy-progesterone.
The estimated incidence of congenital adrenal hyperplasia (CAH) based on different population groups indicates that CAH may occur in one out of every 11,000 live births. Failure of early diagnosis of CAH in the affected newborn may lead to life-threatening adrenal crises during the first few months of life, and in the genetic female there may be a need for sex reassignment if the ambiguity of the external genitalia has led to an incorrect sex assignment.
Furthermore, delayed diagnosis is almost always associated with the acceleration of skeletal maturation ultimately leading to short stature and premature development of secondary sex characteristics in male children and further virilization in female affected children. In view of the described known complications of the unrecognized and untreated disease, and in view of the relatively high frequency of the gene, as reflected in incidence, diagnostic delay is a serious problem.
A rapid screening program for the diagnosis of CAH during the newborn period is therefore desirable.
Until now measurements of urinary 17-ketosteroids and pregnanetriol in the 24-hour urine, and the determination of serum adrenal precursor hormone or androgen hormone have been used as diagnostic tests. In the normal infant, urinary 17-ketosteroid excretion is usually slightly increased during the newborn period. However, pregnanetriol in normal infants and infants with CAH, may not be elevated in urine. Therefore, these tests may not be diagnostic of CAH during the first few days of life. More recently, radioimmunoassay for the measurement of 17.alpha.-OH-progesterone has become available. The usefulness of this assay for the diagnosis of CAH has been well established (see, e.g., Youssefnejadian et al, Clin. Endocrinol. 4; 451, 1975, I. A. Hughes et al, J Pediatr 88: 766, 1976; and B. M. Lippe et al, J Pediatr 85: 782, 1974). However, all the prior art methods for the determination of steroids involve the analysis of blood plasma and require relatively large amounts of blood for the separation of the blood cell mass from the plasma. Further, sample collection by veno puncture is inconvenient in small infants.
In the recently developed radioimmunoassays, radiological means are employed to detect and/or measure the presence of a steroid in the patient's blood (or urine). In these radioimmunoassay tests, a solution of an antibody of the steroid is placed in contact with a mixture of the steroid, which has been extracted from a sample of the patient's body fluid to be tested and a known amount of the same steroid tagged with a radioactive isotope. The steroid in the test sample and the labeled steroid compete for interaction with the steroid antibody. The resulting steroids-antibody-complex is then separated from the fluid and either fraction may be analyzed radiologically in order to determine the respective proportions of the labeled and unlabeled steroid which became bound to the antibody. The concentration of steroid in the sample can be calculated from this information, since the proportion of labeled and unlabeled steroid will be in the same proportion in both fractions. The radioimmunoassay techniques exhibit a high degree of accuracy and specificity. Yet, like other prior art methods for steroid determination, the radioimmunoassay techniques also require relatively large amounts of blood for the preliminary separation of blood plasma from the hematocrit, and also have the usual disadvantage that samples can be stored only at low temperature, since the liquid samples are easily infected and spoiled by growth of bacteria.